Immunosuppression involves reactions that reduce the activation or efficiency of the immune system. Immunosuppressive reactions are either due to intentional medical actions or derivable from a natural background and are, thus, largely pathogenic for the body.
In the first case of intentional medical actions, immunosuppressive activity of compounds is used for the modulation, in particular the controlled and purposeful inhibition or prevention of the activity of the immune system. The corresponding compounds are generally summarized as immunosuppressants or immuno-suppressive drugs. Immunosuppressive drugs are a heterogenic collection including the following groups: glucocorticoids, cytostatics, antibodies, drugs acting on immunophilins, TNF-binding proteins and interferons. Immunosuppressives are used, for example, to prevent the rejection of transplanted organs and tissues (e.g., bone marrow, heart, kidney, liver), treat autoimmune diseases or diseases that are most likely of autoimmune origin (e.g., rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, sarcoidosis, focal segmental glomerulosclerosis, Crohn's disease, Behcet's Disease, pemphigus, and ulcerative colitis) or treat non-autoimmune inflammatory diseases (e.g., long term allergic asthma control).
In the second case of natural background, immunosuppression can occur, for example, in malnutrition, aging, many types of cancer (such as leukemia, lymphoma, multiple myeloma), and certain chronic infections such as acquired immunodeficiency syndrome (AIDS). The unwanted effect of this immunosuppression is immunodeficiency that results in increased susceptibility to pathogens such as bacteria and virus. Moreover many microbial pathogens have evolved, in order to successfully infect a host organism, intriguing mechanisms to subvert host defenses (Sansonetti, 2004). These microbes circumvent and undermine innate and specific host defenses.
One of the most fascinating and widespread pathogenicity modules of Gram-negative pathogens is the type III secretion system (T3SS) that targets essential cytoplasmic processes of the host cell by directly injecting so-called effector proteins into the cytoplasm via a molecular injection machine (‘molecular syringe’) (Cornelis, 2002a; Cornelis, 2002b; Cornelis & Wolf-Watz, 1997). Bacterial effector proteins interfere with signaling mechanisms of the host cells, including those triggering immune responses. Especially pathogenic bacteria of the genus Yersinia, Shigella, or Salmonella harbor a wide range of effector proteins that target signaling mechanisms such as MAPK signaling cascades or pathways leading to repression of NF-kB activation (Matsumoto & Young, 2009). Moreover numerous effector proteins of pathogenic bacteria, which utilize type III or IV secretion systems to deliver effector proteins into host cells, usurp the host ubiquitin pathways (Hicks and Galan, 2010; Angot, 2007).
Ubiquitinylation results in the covalent attachment of ubiquitin to a lysine residue on a target protein. Following the initial conjugation, subsequent ubiquitin molecules can be ligated to one of seven lysines in the previously attached ubiquitin molecule, resulting in polyubiquitinylation of various linkages. Therefore, a substrate can be monoubiquitinylated at a single lysine residue, multi-ubiquitinylated at multiple lysine residues, or polyubiquitinylated at one or more lysine residues. The type of ubiquitinylation and the topology of the ubiquitin chains formed direct substrate fate. Ubiquitinylation can signal for proteasome-dependent degradation or function as non-proteolytic signals important for DNA repair, signal transduction and vesicular trafficking.
Ubiquitinylation involves an enzymatic cascade resulting in the formation of an isopeptide bond between ubiquitin and internal lysine residues of a substrate protein. This process involves an ubiquitin-activating enzyme (E1), which forms a thioester bond between a catalytic cysteine and the carboxy terminal glycine residue of ubiquitin. The ubiquitin is then transferred to an ubiquitin-conjugating enzyme (E2). Finally, an ubiquitin ligase (E3) facilitates the covalent conjugation of ubiquitin from an ubiquitin-loaded E2 to one or more lysine residues in the substrate. Therefore, E3 ubiquitin ligases confer specificity to the reaction through substrate binding. E3 ubiquitin ligases are defined by their ability to facilitate the transfer of ubiquitin from a cognate E2 to a specific substrate. There are two major known types of E3 ubiquitin ligases in eukaryotes, which possess distinct structural and mechanistic properties: the RING (really interesting new gene)/U-box domain and the HECT (homologous to E6-associated protein C terminus) domain. Bacterial effector proteins belonging to these ubiquitin ligase families are e.g. NIeG2-3 from Escherichia coli ssp., LubX from Legionella spp. or SopA from Salmonella ssp (Hicks and Galan, 2010; Angot, 2007). In addition to that, another family of E3 ubiquitin ligases has been described that possesses a structural domain (termed NEL-domain for Novel E3 Ligase), which is distinct from either the RING or HECT domains (see e.g. Hicks and Galan, 2010). NEL E3 ligases comprise a large family of a bacterial effector proteins encoded by pathogenic bacteria, including Shigella ssp., Salmonella ssp., Yersinia spp., Pseudomonas spp., and Escherichia coli ssp. Examples of bacterial NEL E3 Ubiquitin ligases are IpaH1, IpaH1.4, IpaH2, IpaH2.5, IpaH3, IpaH4, IpaH4.5, IpaH5, IpaH6, IpaH7, IpaH7.8, IpaH9.8 (from Shigella spp.); Slrp, SspH1, SspH2 (from Salmonella spp.); YPA_3361, YPA_3364 (from Yersinia spp.); PflO1_4099, PflO1_4565, PP_2212, PP_2394, PSPTO_1492, PSPTO_4093 (from Pseudomonas spp.); EcoI5_01000486, EcoI5_01001536, EcoI5_01001967, EcoI5_01003958, EcoI5_01004202, EcoI5_01004539, EcoI5_01004764, EcoI5_01004830, and EcoI5_01004885 (from Escherichia coli spp.) (Hicks and Galan, 2010). Several of these bacterial NEL E3 ligases comprise an N-terminal leucine-rich repeat (LRR) domain and are therefore also classified as leucine-rich repeat (LRR) proteins. If said LRR is a LRR of the LPX-subtype, the proteins are called effector proteins of the LPX-subtype or effector proteins of LPX-family (Miao et al, 1999). Examples of effector proteins which comprise a NEL-domain and belong to the LPX-subtype are SspH1, SspH2, SlrP, IpaH4.5, IpaH7.8, and IpaH9.8.
For T3SS-dependent translocation of effector proteins, bacterial attachment to target cells is essential. Only cells that are contacted directly are infected via the T3SS injection machine. Surprisingly, it was recently shown that the Yersinia protein YopM, a secreted effector protein, is able to translocate into eukaryotic cells independently of the T3SS. Besides a T3SS-dependent translocation, YopM is able to autonomously penetrate the eukaryotic cell membrane and integrate into the cell cytosol (Riter et al, 2010; Scharnert et al, 2013). It was suggested that the N-terminal α-helices of YopM mediate autonomous cell-penetration. Moreover it was shown that YopM can thereby deliver heterologous cargos into eukaryotic cells. Furthermore it was shown that YopM down-regulates the transcription of pro-inflammatory cytokines (e.g. TNFα, IL-12, IL-15, and IL-18) after autonomous penetration into host cells (Rüter et al, 2010).
Cell-penetrating peptides (cell-penetrating proteins, cell-permeable protein, CPPs) are proteins that are able to cross the cell membrane on their own. CPPs such as the trans-activator of transcription (Tat) protein encoded by the human immunodeficiency virus type I (HIV-1) are usually relatively short proteins or peptides (5-40 amino acids) with the ability to enter cells by different mechanisms (Frankel & Pabo, 1988) (Green & Loewenstein, 1988). Since the early studies on CPPs, numerous natural and synthetic peptides have been described to penetrate eukaryotic plasma membranes and deliver heterogeneous cargos into the host cell (Langel, 2011). The uptake mechanisms of most CPPs are poorly understood. Various uptake mechanisms of CPPs are discussed in the literature. It appears that different mechanisms can be involved in uptake of the diverse CPPs rather than a general mechanism. Initial binding of CPP to the plasma membrane seems to depend on electrostatic interactions of positively charged amino acids with negatively charged plasma membrane components. Two different models are currently discussed describing potential uptake mechanisms that follow the initial binding of the CPP to the plasma membrane. CPP uptake might be mediated by different endocytic uptake mechanisms including macropinocytosis, Clathrin-dependent and independent endocytosis, and Caveolae-dependent endocytosis, wherein after uptake, CPP need to escape from endosomal compartments during intra-cellular transport. Moreover internalization of the CPPs might occur by direct membrane penetration mechanisms, including inverted micelle and pore formation (Trabulo et al, 2010). CPPs have the ability to cross cellular membranes, either alone or in association with cargo molecules.
YopM was the first identified bacterial CPP and opened the class of bacteria-derived CPPs within the heterogeneous group of CPPs (Rüter et al, 2010). YopM shares the ability of known CPPs to translocate across eukaryotic plasma membranes and it has the capacity to deliver molecular cargos such as GFP intracellularly. However, YopM does not show sequence homologies to known CPPs, indicating unique features that promote the uptake of YopM.
Crossing the plasma membrane is a prerequisite for intracellular targeted drug and/or compound delivery (for example in gene therapy where the gene/nucleic acid has to be delivered to an intracellular compartment). Cell penetrating peptides (CPPs) are known to transport cargo molecules attached to them into cells primarily by endocytosis. Nevertheless, there is an ongoing need in the art to provide compounds, which are able to cross the plasma membrane of higher cells. Especially in the field of immunotherapy, there is a great need for improved strategies for delivering immunomodulatory agents into cells. Moreover, self-delivering immunomodulatory agents would greatly improve the current immunotherapeutic strategies.
Although the above-mentioned immunosuppressive drugs are valuable medical tools, they are not without side effects and risks. Because the majority of them act non-selectively, the immune system is less able to resist infections and the spread of malignant cells. Furthermore, the production of the majority of immunomodulatory drugs is time-consuming and expensive. Therefore, there is a need for the provision of new, effective, cell-specific, selectively acting, and inexpensive immunomodulators, particularly immunosuppressants, preferably with lower side effects and risks.
The solution to the above-indicated technical problem is achieved by providing the embodiments as characterized herein and summarized below.